Assessment of oxidant stress in vitro and in vivo

ABSTRACT

There is provided a method of assessing oxidant stress by measuring polymerization of proteins. Also provided is a marker for oxidant stress which includes a polymerized protein. A kit for use in assessing oxidant stress, the kit including an assay for detecting polymerized proteins is also provided. A method of lowering oxidant stress by administering to a patient an effective amount of at least one reducing agent is also provided. A pharmaceutical composition for lowering oxidant stress, the pharmaceutical having an effective amount of reducing agent and a pharmaceutically acceptable carrier is also provided.

CROSS-RELATED REFERENCE SECTION

[0001] This application claims the benefit of priority under 35 U.S.C.Section 119(e) of United States Provisional Patent Application No.60/221,631, filed Jul. 28, 2000, which is incorporated herein byreference.

GOVERNMENT SUPPORT

[0002] Research in this application was supported in part by a contractfrom National Institute of Environmental Health Sciences (N43 ES 95438).The government has certain rights in the invention.

BACKGROUND OF INVENTION

[0003] 1. Field of the Invention

[0004] The present invention generally relates to methods of assessingand preventing oxidant stress in vitro and in vivo. More specifically,the present invention relates to methods of measuring nitration andpolymerization of proteins which can be used for assessing oxidantstress and methods of preventing nitration and/or polymerization ofprotein due to oxidant stress.

[0005] 2. Description of Related Art

[0006] It is generally known in the art that superoxide is producedunder oxidant stress. This reactive molecule directly or indirectlyreacts with macromolecules such as DNAs and proteins and depletesreducing agents such as glutathione in the cells.

[0007] Prostaglandin H₂ synthases, which are membrane-bound enzymes,catalyze the committed step in the biosynthesis of the prostaglandinsand thromboxanes. Prostaglandin H₂ synthases have cyclooxygenase andperoxidase activities (1). Prostaglandin H₂ synthases add molecularoxygen to arachidonic acid to form prostaglandin G₂. Prostaglandin G₂ isthen rapidly converted to prostaglandin H₂ by reduction of the peroxideto a hydroxyl group. Prostaglandin H₂ is enzymatically ornon-enzymatically converted to prostaglandin F_(2α), prostaglandin E₂,prostaglandin D₂, prostaglandin I₂ (prostacyclin) or thromboxane A₂(2-4). In addition to constitutively expressed prostaglandin H₂ synthaseform 1, a second inducible form of prostaglandin H₂ synthase,prostaglandin H₂ synthase form 2, was discovered in chicken fibroblasts(5) and murine 3T3 cells (6). Prostaglandin H₂ synthase form 2 issimilar to prostaglandin H₂ synthase form 1 with regard to molecularsize, subunit composition, and general reaction mechanism. The twoprostaglandin H₂ synthase isoforms have 60% identity in their primarysequences (7).

[0008] Prostaglandin biosynthesis is the target for non-steroidalanti-inflammatory drugs (NSAIDs). Prostaglandin H₂ synthase is theprimary target of aspirin, indomethacin, ibuprofen, and other NSAIDs(5,8). Increased expression of both inducible nitric oxide (NO) synthaseand prostaglandin H₂ synthase form 2 has been reported in intestinalinflammation (9-11).

[0009] Cytochrome c induces apoptosis by translocation from themitochondrial membrane to the cytoplasm followed by binding to theapoptotic protease activating factor-1 (Apaf-1) which activates caspases(12). Nitration and/or polymerization of cytochrome c in mitochondriamay compromise its translocation and/or its caspase activation function.

[0010] Nitration of several amino acid residues of proteins is a resultof the reaction of NO with superoxide which is produced under oxidantstress to form peroxynitrite, a potent toxic oxidant. Recently, it wasreported that nitrotyrosine was also formed via a tyrosyl radicalproduced during catalysis of prostaglandin H₂ synthase form 2 (13).Nitration of caspase-3 (14), ribonucleotide reductase (15), andcytochrome P450 2B1 (16) resulted in loss of catalytic activity.Interestingly, nitration of cysteine residue of ovine prostaglandin H₂synthase form 1 enhanced catalytic activity by alteration of secondarystructure of the enzyme (17).

[0011] While it is well known in the art that oxidant stress can lead tofurther problems within the body which, as set forth above, there are nomethods or products which limit or eliminate oxidant stress. Theseproblems can include cancer and other ailments. Accordingly, it isdesirable to develop new methods and markers for determining oxidantstress.

SUMMARY OF THE INVENTION

[0012] According to the present invention, there is provided a method ofassessing oxidant stress by measuring polymerization of proteins. Alsoprovided is a marker for oxidant stress which includes a polymerizedprotein. A kit for use in assessing oxidant stress, the kit including anassay for detecting polymerized proteins is also provided. A method oflowering oxidant stress by administering to a patient an effectiveamount of at least one reducing agent is also provided. A pharmaceuticalcomposition for lowering oxidant stress, the pharmaceutical having aneffective amount of reducing agent and a pharmaceutically acceptablecarrier is also provided.

DESCRIPTION OF THE DRAWINGS

[0013] Other advantages of the present invention will be readilyappreciated as the same becomes better understood by reference to thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

[0014] FIGS. 1A-C shows the concentration-dependent nitration ofcytochrome c by peroxynitrite (PN); Panel A, in lanes 1 and 2, showsSDS-PAGE analysis of 2 μg of cytochrome c obtained after incubation with0.5 mM PN and deactivated PN, respectively; Panel B shows thatcytochrome c was incubated with various concentrations of PN (0.032 to0.13 mM); each lane contains 0.5 μg of cytochrome c; Panel C showsanalysis of Western blot analysis shown in FIG. 1, Panel B and blotsfrom two other experiments; bands were quantitated using a MolecularDynamics Personal Densitometer; Western blot analysis was carried outwith polyclonal antibodies produced against nitrotyrosine using thealkaline phosphatases system;

[0015]FIG. 1D and E show quantitation of nitrotyrosine inperoxynitrite-treated cytochrome c; Panel D shows Western blot analysisof peroxynitrite-treated cytochrome c and tetranitromethane-treatedbovine serum albumin (BSA); Panel E shows the amount of nitrotyrosineper pmol of cytochrome c at each of the peroxynitrite concentrations;quantitation of cytochrome c content in peroxynitrite-treated cytochromec was determined with nitrated BSA as standard.

[0016]FIG. 2 shows the effect of arachidonic acid (AA) metabolism ofprostaglandin H₂ synthase form 2 (PGHS-2) on nitration of cytochrome c,the reaction mixture contained cytochrome c, 40 μg, ovine PGHS-2, 104units, AA, 590 μM, epinephrine, 31 μg, hematin, 1 μM, anddiethylenetetraamine (DETA) NONOate (NO donor), 300 μM, as indicated inthe figure; the reaction mixture in 100 mM phosphate buffer with a pH of7.4 (total volume, 200 μl) was incubated at 37° C. for 30 minutes; eachlane contained 8 μl of reaction mixture; proteins were separated bySDS-PAGE. Western blot analysis was carried out with monoclonalantibodies produced against nitrotyrosine using the alkaline phosphatasesystem;

[0017]FIG. 3 Panels A and B show the inhibition of nitration ofprostaglandin H₂ synthase form 2 (PGHS-2) by addition of glutathione(GSH); nitration was induced by treatment of peroxynitrite (PN); lane 1included 16 μg of sheep PGHS-2 without treatment; lanes 2 and 3 includedPGHS-2 with 450 μM of PN and 31 μM of GSH treatment; and lanes 4 and 5included PGHS-2 with PN treatment; samples were denatured withβ-mercaptoethanol (β-ME) contained in the loading buffer were analyzedby SDS-PAGE; the proteins were electroblotted to a nitrocellulosemembrane and Western blot analysis was carried out using monoclonalantibodies against nitrotyrosine with an alkaline phosphatase system;

[0018]FIG. 4 shows the effect on catalytic activities of prostaglandinH₂ synthases form 1 (PGHS-2) and form 2 (PGHS-2) after incubation withperoxynitrite (PN); either sheep PGHS-2 or PGHS-2 was treated with PNprior to measuring the metabolic activity of the enzymes; activity wasdetermined by conversion of ¹⁴C-arachidonic acid (AA) to its metabolitesand visualized by thin layer chromatography (TLC);

[0019]FIGS. 5A and B, show dimerization of prostaglandin H₂ synthaseform 2 (PGHS-2) via a cysteine disulfide-bond following treatment withperoxynitrite (PN). Lane 1 included 16 μg of sheep PGHS-2 withouttreatment; lanes 2 and 3 included PGHS-2 with 450 μM of PN and 31 μM ofglutathione treatment; and lanes 4 and 5 included PGHS-2 with PNtreatment; samples were analyzed by SDS-PAGE without β-mercaptoethanolin the loading buffer; proteins were electroblotted to a nitrocellulosemembrane and Western blot analysis was carried out using eitherantibodies against nitrotyrosine (Panel A) or antibodies against PGHS-2(Panels B-1 and B-2) with a horseradish peroxidase-ECL (Panel B-1) oralkaline phosphatase-ECL (Panel B-2) system; and

[0020]FIGS. 6A and B, show prostaglandin H₂ synthase form 2 (PGHS-2)nitrated and dimerized in vivo; Panel A shows sheep PGHS-2 (16 μg)analyzed by SDS-PAGE with β-mercaptoethanol (+βME) in the loadingbuffer; Panel B shows PGHS-2 (16 μg) without treatment analyzed bySDS-PAGE without β-mercaptoethanol (−βME) in the loading buffer; theproteins were electroblotted to a nitrocellulose membrane and Westernblot analysis was carried out using either antibodies againstnitrotyrosine (Panel A and Panel B-3) or antibodies against PGHS-2(Panel B-2), coomassie blue staining of the PGHS-2 after SDS-PAGE wascarried out without this addition of β-ME (−βME) in the loading bufferis shown in Panel B-2.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention generally provides a method to assessoxidant stress by measuring polymerized proteins. The polymerizedproteins can be assessed by conventional detection methods includingelectrophoresis, chromatography, or Western blot analysis using antibodyagainst the protein or nitrated amino acid residues.

[0022] The present invention also provides a method to assess oxidantstress by measuring nitrated and polymerized proteins. The nitrated andpolymerized proteins can be separated by conventional methods includingelectrophoresis and chromatography and detected by Western blot analysisusing antibodies against nitrated amino acid residues. The methodfurther includes assessing oxidant stress by measuring formation ofprotein polymers connected by disulfide bonds. The methods of thepresent invention include methods to assess oxidant stress by measuringpolymerized cytochrome c and prostaglandin H₂ synthases induced byoxidants such as peroxynitrite, a potent toxic oxidant. Peroxynitrite isformed as a result of reaction of NO with superoxide produced underoxidant stress.

[0023] The method includes assessing oxidant stress by measuringcytochrome c nitrated by oxidants such as peroxynitrite.

[0024] Oxidant stress in biological systems disrupts cellular structuresand functions, and contributes to disease development. By assessingoxidant stress, it is meant that the present inventive assay is capableof being an indication of oxidant stress in vitro and in vivo. Incombination with conventional detection methods, the assay indicates arelationship between oxidant stress and proteins nitrated and/orpolymerized via disulfide or non-disulfide bonds.

[0025] Previously no studies have been reported on oxidantstress-induced polymerization and especially simultaneous nitration andpolymerization of a protein. Moreover polymerization of proteins byoxidant stress via sulfur hydryl group of cysteine residues andprevention of the polymerization by addition of a reducing agent has notbeen reported. Existence of the disulfide boned dimeric form ofprostaglandin H₂ synthases is therefore unexpected.

[0026] Polymerization of cytochrome c after peroxynitrite treatmentoccurs via non-disulfide bonds. Nitration and polymerization ofcytochrome c after peroxynitrite treatment is dose-dependent (FIG. 1).Cytochrome c was nitrated and polymerized when peroxynitrite wasreplaced with NO donor (DETA NONOate) in the presence of peroxideproduced by prostaglandin H₂ synthase during catalysis of arachidonicacid (FIG. 2). Existence of a nitrated species with a molecular mass of30 or 45 kDa species was indicative of oxidant stress.

[0027] Prostaglandin H₂ synthase form 2, isolated from sheep placenta,contained nitrated tyrosine residues (FIGS. 2 and 6). The nativeprostaglandin H₂ synthase form 2 was found to be dimerized via acysteine-disulfide bond. The prostaglandin H₂ synthase form 2 dimercontained higher levels of nitrated tyrosine residues compared with themonomer (FIG. 6). The in vivo nitration and dimerization ofprostaglandin H₂ synthase form 2 was a result of oxidant stress mediatedby prostaglandin H₂ synthase and NO synthase. Nitration andpolymerization of prostaglandin H₂ synthase was not an artifact whichoccurred during purification of the enzyme considering this extremelyunstable enzyme maintained its activity.

[0028] Dimerization of prostaglandin H₂ synthase via cysteine disulfidebond also occurred in vitro. Prostaglandin H₂ synthase was dimerized viacysteine-disulfide bond after peroxynitrite treatment and theprostaglandin H₂ synthase dimer contained higher levels of nitratedtyrosine residues compared with the monomer (FIG. 6).

[0029] In the previously reported experiments, prostaglandin H₂ synthasewas treated with β-mercaptoethanol to break disulfide bonds prior toseparation of the prostaglandin H₂ synthase by electrophoresis. Breakageof disulfide bonds by addition of β-mercaptoethanol preventedresearchers determining that prostaglandin synthase was polymerized bydisulfide bonds by oxidant insults in vitro or in vivo.

[0030] The present experiments also demonstrated that nitration anddimerization of prostaglandin H₂ synthase was prevented by the additionof a reducing agent such as GSH (FIGS. 3,5,6). The sulfur hydryl groupof GSH quenched activity of toxic oxidants such as peroxynitrite andprevented nitration and dimerization of prostaglandin H₂ synthase. Ithas been reported that peroxynitrite nitrated amino acid residues ofproteins (15,17,18). Thus, it was hypothesized that GSH would benitrated and form a nitrated form of glutathione, GS-NO. However, whenthe reaction mixture was analyzed by HPLC and the peaks were identifiedusing authentic standards, it was found that no GS-NO was formed.Instead GSH was oxidized to GS-SG, which is a dimerized form ofglutathione. This reaction can therefore be utilized to measure theoxidizing power of oxidizing molecules, such as peroxynitrite bymeasuring the amount of GS-SG formed from GSH. Additionally, a methodfor using the s-dimerized form of chemical can also include using theformation of an s-dimerized chemical to assess the quenching power ofreducing agents, such as antioxidants, against the oxidizing power ofmolecules, such as peroxynitrite.

[0031] The results of the experiment also demonstrated that nitrationand polymerization of cytochrome c and prostaglandin H₂ synthase bynon-disulfide and disulfide bonds, respectively, occurred in vivo and invitro and that quenching of the nitration and polymerization by additionof GSH were unexpected.

[0032] Polymerization can be assessed by visualization of proteinsseparated by differences in the molecular mass of the polymerizedproteins. Separation of polymerized proteins from their monomer can beachieved by SDS-gel or gel filtration chromatography. Nitration can beassessed by Western blot analysis using antibodies against nitratedamino acid residues or by GC/MS of the nitrated amino acid residues.

[0033] In general, the assessment of the sample is done utilizing sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) withWestern blot analysis as described in the Examples set forth herein.However, alternative electrophoresis, such as electrophoresis withWestern blot analysis or chromatography can be used. Most of thetechniques used in performing SDS-PAGE followed by Western blot analysisare widely practiced in the art, and most practitioners are familiarwith the standard resource materials which describe specific conditionsand procedures as set forth below.

[0034] In general, sample treatment with gel loading buffer with orwithout β-mercaptoethanol and SDS-PAGE followed by Western blot analysisis employed to assess the status of polymerization and/or nitration ofproteins in a specimen. SDS-PAGE followed by Western blot analysis iswell known to those skilled in the art.

[0035] Available assays that separate polymers from monomers areextensively described in the patent and scientific literature, which canbe adapted to be used with the methods of the present invention.

[0036] The polymerized cytochrome c and prostaglandin H₂ synthase aremeasured utilizing the SDS-PAGE followed by Western blot analysis, withCoomassie blue stained gel and with antibodies which recognized nitratedtyrosine, cytochrome c and prostaglandin H₂ synthase.

[0037] The present invention also provides a marker and kit for use indetermining oxidant stress. The marker is preferably a polymerizedprotein. Alternatively, the marker can be a nitrated polymerizedprotein. In an embodiment, the marker is selected from the group ofpolymerized prostaglandin H₂ synthase, nitrated-polymerizedprostaglandin H₂ synthase, polymerized cytochrome c, nitrated,polymerized cytochrome c, 30 kDa cytochrome c, nitrated 30 kDacytochrome c, 45 kDa cytochrome c, and nitrated 45 kDa cytchrome c.Alternatively, the marker can be a disulfide bonded polymerized proteinor nitrated disulfide bonded polymerized protein. In these embodiments,the markers are preferably selected from the group consistingessentially of disulfide bonded polymers of prostaglandin H₂ synthaseand nitrated, disulfide bonded polymers of prostaglandin H₂ synthase.

[0038] As stated above, the marker can be used in conjunction with a kitfor assessing oxidant stress. The kit includes an assay for detectingthe polymerized proteins or the nitrated polymerized proteins. The assaycan be any of the assays listed above or as any other assays which areknown to those of skill in the art to be useful in the kit. The assayrequires a detecting device for detecting either the polymerizedproteins, nitrated polymerized proteins, the formation of disulfidebonded polymerized proteins, or the formation of nitrated disulfidebonded polymerized proteins.

[0039] Further, the present invention provides a method for loweringoxidant stress by adding an effective amount of a reducing agent. Areducing agent prevents the polymerization, nitration or formation ofdisulfide bonded prostaglandin H₂ synthase dimers. This reducing agent,or antioxidant, can be any reducing agent known to those of skill in theart to have the capability of preventing the polymerization of proteins.In the preferred embodiment, the reducing agent is glutathione. However,this is not meant to be limiting but is instead included herein as anexample of one reducing agent which is able to use in conjunction withthe methods of the present invention. Other examples of reducing agentsinclude glutathione monoethyl ester (GSH precursor), cysteine,methionine and sulfur containing chemicals.

[0040] Also provided by the present invention is a pharmaceuticalcomposition for use in lowering oxidant stress. The pharmaceuticalincludes at least one reducing agent and a pharmaceutically acceptablecarrier. The reducing agent, or antioxidant, can be any reducing agentwhich is able to prevent the polymerization of proteins, nitration ofproteins or the nitrated disulfide prostaglandin H₂ synthase dimersformation. In the preferred embodiment the reducing agent isglutathione. However, as set forth above, the inclusion of glutathioneis intended only for illustrative purposes and not as a limitation.Accordingly, any other reducing agents which are capable of performingin the above indicated manner can also be used without departing fromthe spirit of the present invention.

[0041] The above discussion provides a factual basis for the method ofthe present invention to measure nitrated and/or polymerized proteins asan assessment of oxidant stress in vitro and in vivo. The methods usedwith and the utility of the present invention can be shown by thefollowing non-limiting examples and accompanying figures.

EXAMPLES Materials and Methods Materials

[0042] Sheep prostaglandin H₂ synthases form 1 and 2, DETA NONOate (NOdonor) and antibodies against nitrotyrosine and prostaglandin H₂synthase were obtained from Cayman Co. (Ann Arbor, Mich.). Goatanti-rabbit immunoglobulin G (IgG) and horseradish peroxidase- andalkaline phosphatase-conjugated donkey anti-rabbit IgG were purchasedfrom Jakson ImmunoResearch Laboratories, Inc. (West Grove, Pa.).Arachidonic acid was obtained from Biomol Research Lab (PlymouthMeeting, Pa.). [¹⁴C] arachidonic acid was obtained from NEN Life ScienceProducts, Inc. (Boston, Mass.) (specific activity 53 mCi/mmol).Calorimetric substrates of alkaline phosphatase were obtained fromBioRad Laboratories (Hercules, Calif.). Electrochemiluminescentsubstrates of horseradish peroxidase and alkaline phosphatase wereobtained from BioRad Laboratories and Amersham Pharmacia Biotech(Piscataway, N.J.), respectively.

[0043] Other reagents were obtained from Sigma Chemical Co.

Sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis (SDS-PAGE) andWestern Blot Analysis

[0044] SDS-PAGE was carried out on 10% or 15% gels, according to Laemmli(18), with or without the addition of β-mercaptoethanol in gel loadingbuffer before heat denaturization of proteins. The separated proteinswere electroblotted onto a cellulose membrane and Western blot analyseswere carried out using alkaline phosphatase system as previouslydescribed (19,20). Visualization of bands was accomplished by incubationwith a mixture of 5-bromo-4-chloro-3-indolylphosphatate p-toluidine andnitrobluetetrazolium. Western blot analyses were also carried out usinghorseradish peroxidase or alkaline phosphatase system withelectrochemiluminescent substrates.

Prostaglandin H₂ Synthase Activity Analysis Using Thin LayerChromatography (TLC)

[0045] Prostaglandin H₂ synthase activity analysis of sheepprostaglandin H₂ synthases form 1 and 2 obtained from ram seminalvesicle and placenta, respectively, were carried out as previouslydescribed (21) with or without pretreatment with peroxynitrite. Briefly,purified or partly purified sheep prostaglandin H₂ synthases form 1 (9units) and form 2 (12 units) were pretreated with or withoutperoxynitrite at 37° C. for 30 minutes. [¹⁴C]arachidonic acid (AA) (25μM) (NEN, specific activity 53 mCi/mmol) was added to the prostaglandinH₂ synthase form 1 or form 2 in 0.2 ml of PBS, and incubated at 37° C.for 10 minutes. Prostaglandin H₂ synthase metabolites in the medium wereextracted with ethyl acetate, dried down in a centrifugal evaporator,spotted on a silica TLC plate and separated using A-9 solvent (ethylacetate : trimethylpentane: acetic acid : water, 55:25:10:50). The TLCassay measured products resulting from both the cyclooxygenase andperoxidase activities of prostaglandin H₂ synthase. The immediateproduct of the prostaglandin H₂ synthase reaction, prostaglandin H₂, wasunstable and broke down in a non-enzymatic fashion to a variety ofprostaglandins which were detectable by TLC. The Rf pattern in the A-9solvent system of the break down products of prostaglandin H₂ producedby ram seminal vesicle prostaglandin H₂ synthase form 1 was wellcharacterized. Thus, prostaglandin E₂, prostaglandin D₂ andprostaglandin F_(2α) formed by prostaglandin H₂ synthase form 2 wereidentified by comparing these metabolites with metabolites formed bysheep prostaglandin H₂ synthase form 1.

Delivery of Gene Products/therapeutics (Compound)

[0046] The compound of the present invention is administered and dosedin accordance with good medical practice, taking into account theclinical condition of the individual patient, the site and method ofadministration, scheduling of administration, patient age, sex, bodyweight and other factors known to medical practitioners. Thepharmaceutically “effective amount” for purposes herein is thusdetermined by such considerations as are known in the art. The amountmust be effective to achieve improvement including but not limited toimproved survival rate or more rapid recovery, or improvement orelimination of symptoms and other indicators as are selected asappropriate measures by those skilled in the art.

[0047] In the method of the present invention, the compound of thepresent invention can be administered in various ways. It should benoted that it can be administered as the compound or as pharmaceuticallyacceptable salt and can be administered alone or as an active ingredientin combination with pharmaceutically acceptable carriers, diluents,adjuvants and vehicles. The compounds can be administered orally,subcutaneously or parenterally including intravenous, intraarterial,intramuscular, intraperitoneally, and intranasal administration as wellas intrathecal and infusion techniques. Implants of the compounds arealso useful. The patient being treated is a warm-blooded animal and, inparticular, mammals including man. The pharmaceutically acceptablecarriers, diluents, adjuvants and vehicles as well as implant carriersgenerally refer to inert, non-toxic solid or liquid fillers, diluents orencapsulating material not reacting with the active ingredients of theinvention.

[0048] It is noted that humans are treated generally longer than themice or other experimental animals exemplified herein which treatmenthas a length proportional to the length of the disease process and drugeffectiveness. The doses may be single doses or multiple doses over aperiod of several days, but single doses are preferred.

[0049] The doses may be single doses or multiple doses over a period ofseveral days. The treatment generally has a length proportional to thelength of the disease process and drug effectiveness and the patientspecies being treated.

[0050] When administering the compound of the present inventionparenterally, it will generally be formulated in a unit dosageinjectable form (solution, suspension, emulsion). The pharmaceuticalformulations suitable for injection include sterile aqueous solutions ordispersions and sterile powders for reconstitution into sterileinjectable solutions or dispersions. The carrier can be a solvent ordispersing medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, liquid polyethylene glycol, and thelike), suitable mixtures thereof, and vegetable oils.

[0051] Proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Nonaqueousvehicles such a cottonseed oil, sesame oil, olive oil, soybean oil, cornoil, sunflower oil, or peanut oil and esters, such as isopropylmyristate, may also be used as solvent systems for compoundcompositions. Additionally, various additives which enhance thestability, sterility, and isotonicity of the compositions, includingantimicrobial preservatives, antioxidants, chelating agents, andbuffers, can be added. Prevention of the action of microorganisms can beensured by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption of theinjectable pharmaceutical form can be brought about by the use of agentsdelaying absorption, for example, aluminum monostearate and gelatin.According to the present invention, however, any vehicle, diluent, oradditive used would have to be compatible with the compounds.

[0052] Sterile injectable solutions can be prepared by incorporating thecompounds utilized in practicing the present invention in the requiredamount of the appropriate solvent with various of the other ingredients,as desired.

[0053] A pharmacological formulation of the present invention can beadministered to the patient in an injectable formulation containing anycompatible carrier, such as various vehicle, adjuvants, additives, anddiluents; or the compounds utilized in the present invention can beadministered parenterally to the patient in the form of slow-releasesubcutaneous implants or targeted delivery systems such as monoclonalantibodies, vectored delivery, iontophoretic, polymer matrices,liposomes, and microspheres. Examples of delivery systems useful in thepresent invention include: U.S Pat. Nos. 5,225,182; 5,169,383;5,167,616; 4,959,217; 4,925,678; 4,487,603; 4,486,194; 4,447,233;4,447,224; 4,439,196; and 4,475,196. Many other such implants, deliverysystems, and modules are well known to those skilled in the art.

[0054] A pharmacological formulation of the compound utilized in thepresent invention can be administered orally to the patient.Conventional methods such as administering the compounds in tablets,suspensions, solutions, emulsions, capsules, powders, syrups and thelike are usable. Known techniques which deliver it orally orintravenously and retain the biological activity are preferred.

[0055] In one embodiment, the compound of the present invention can beadministered initially by intravenous injection to bring blood levels toa suitable level. The patient's levels are then maintained by an oraldosage form, although other forms of administration, dependent upon thepatient's condition and as indicated above, can be used. The quantity tobe administered will vary for the patient being treated and will varyfrom about 100 ng/kg of body weight to 100 mg/kg of body weight per dayand preferably will be from 10 mg/kg to 10 mg/kg per day.

Example 1 Nitrated, Polymerized Cytochrome

[0056] Cytochrome c (40 μg) was incubated with 0.5 mM (FIG. 1, Panel A,lane 1) of deactivated (a negative control, FIG. 1, Panel A, lane 2)peroxynitrite in 100 mM phosphate buffer, pH 7.4. Deactivatedperoxynitrite was produced by incubation of 0.5 mM peroxynitrite inbuffer for 5 minutes prior to addition to the reaction mixture.

[0057] After reaction with peroxynitrite or deactivated peroxynitrite, 2μg of the cytochrome c was separated by SDS-PAGE and electroblotted to acellulose membrane. Western blot analysis of the electroblotted proteinswas carried out with polyclonal antibodies produced againstnitrotyrosine using the alkaline phosphatase system. Cytochrome c (14kDa) treated with peroxynitrite was nitrated and polymerized to 30 kDa,45 kDa and higher molecular mass (MM) whereas cytochrome c treated withdeactivated peroxynitrite failed to be nitrated (FIG. 1, Panel A). Thepolymerized cytochrome c showed molecular masses of 30 kDa, 45 kDa and ahigher molecular mass.

[0058] Cytochrome c (40 μg) was incubated with increasing concentrations(0.032 to 0.13 mM) (FIG. 1, Panel B) of peroxynitrite in 100 mMphosphate buffer, pH 7.4. Cytochrome c (0.5 pg) was separated bySDS-PAGE and electroblotted to a cellulose membrane. Western blotanalysis of the electroblotted proteins was carried out with polyclonalantibodies produced against nitrotyrosine using the alkaline phosphatasesystem (FIG. 1, Panel B). The cytochrome c monomer band (14 kDa) shownin Western blot analysis in FIG. 1, Panel B and those obtained from twoother experiments were quantitated using Molecular Dynamics PersonalDensitometer (FIG. 1, Panel C). This result demonstrates that cytochromec treated with peroxynitrite was nitrated and polymerized in adose-dependent manner.

[0059] Peroxynitrite-treated cytochrome c and tetranitromethane-treatedbovine serum albumin (BSA) were separated by 15% SDS-PAGE and analyzedby Western blot analysis using anti-nitrotyrosine IgG (FIG. 1, Panel D).The tetranitromethane-treated BSA was prepared as previously described(22). The amount of nitrotyrosine per pmol of cytochrome c at each ofthe peroxynitrite concentrations was calculated on the basis of thenitrated BSA standard curve following quantitation of cytochrome c band(14 kDa) using a Molecular Dynamics Personal Densitometer (FIG. 1, PanalE).

[0060] The amino acid composition of cytochrome c analyzed by HPLC afteracid hydrolysis revealed that cytochrome c contained tyrosine residues.Peroxynitrite treatment of the cytochrome c decreased the tyrosine peakin a chromatogram of the HPLC. Both cysteine residues in cytochrome care not available for nitration because they are covalently bound to thehomo prosthetic group. Thus, nitration of cytochrome c withperoxynitrite results in nitration of tyrosine residues but not cysteineresidues.

Example 2 Nitrated Cytochrome C (14 kDa), 30 kDa Species and 45 kDaSpecies Produced by Oxidant Stress Mediated by Prostaglandin H₂ Synthase

[0061] Cytochrome c was nitrated and polymerized by peroxynitritetreatment (FIG. 1) as discussed in EXAMPLE 1.

[0062] Reaction mixture in 100 mM phosphate buffer, pH 7.4, (totalvolume, 200 μl) was incubated at 37° C. for 30 minutes. The reactionmixture contained a combination of cytochrome c (40 μg), ovineprostaglandin H₂ synthase form 2 (104 units), arachidonic acid (590 μM),epinephrine (31 μg), hematin (1 μM), and DETA NONOate, an NO donor, (300μM) as indicated in FIG. 2. After incubation, the proteins wereseparated by SDS-PAGE followed by Western blot analysis with an alkalinephosphatase system using monoclonal antibodies produced againstnitrotyrosine.

[0063] By this experiment, it was found that cytochrome c could benitrated and polymerized when peroxynitrite was replaced with a NO donor(DETA NONOate) in presence of prostaglandin H₂ synthase in anarachidonic acid-dependent manner (FIG. 2). This result suggested thatnitration of cytochrome c, the 30 kDa species and 45 kDa species, was aresult of transformation of NO to a reactive molecule, e.g.peroxynitrite, during prostaglandin H₂ synthase-dependent metabolism ofarachidonic acid. Triplet bands of prostaglandin H₂ synthase form 2 inWestern blot analysis were a result of the specific interaction ofnitrotyrosine residues of prostaglandin H₂ synthase with antibodiesagainst nitrotyrosine.

Example 3 Nitrated and Polymerized Prostaglandin H₂ Synthase Produced inVitro and in Vivo and Inhibition of Nitration and Polymerization byAddition of a Reducing Agent

[0064] Treatment of prostaglandin H₂ synthase form 2 with peroxynitriteincreased nitration of tyrosine residues (FIG. 3, Panel A, lanes 4 and5). Contrary to the previously reported results that nitration of acysteine residue of ovine prostaglandin H₂ synthase form 1 enhancedcatalytic activity by alteration of secondary structure of the enzyme(13), nitration induced by peroxynitrite treatment abolished thecatalytic activities of both prostaglandin synthases form 1 and 2 (FIG.4).

[0065] Addition of glutathione (GSH) inhibited the nitration of tyrosineresidues of prostaglandin H₂ synthase form 2 (FIG. 3, Panel A, lanes 2and 3). HPLC analysis of the reaction mixture containing GSH afterperoxynitrite treatment revealed a decrease in the GSH level and aconcurrent increase in the oxidized glutathione (GS-SG) level inaddition to the appearance of an unidentified peak. Nitrated glutathionewhich was predicted to be formed after peroxynitrate treatement,nitrosoglutathione (GS-NO), (see FIG. 3, Panel B) was ruled out as apossible candidate for the unidentified product based on HPLC analysis.

[0066] Whether prostaglandin H₂ synthase was dimerized afterperoxynitrite treatment via a cysteine-disulfide bond was determinedusing SDS-PAGE without addition of β-mercaptoethanol (βME) in the gelloading buffer (FIG. 5, Panels A and B-2 and B-2, lanes 4 and 5). Theperoxynitrite-treated samples migrated both as a monomer (70 kDa) and adimer (140 kDa). This experiment revealed that prostaglandin H₂ synthaseform 2 was dimerized via a disulfide bond.

[0067] Prostaglandin H₂ synthase form 2 samples treated withperoxynitrite in the presence of GSH ran as a monomer (FIG. 5, PanelsB-2 and B-2, lanes 2 and 3) which was not nitrated (FIG. 5, Panel A,lanes 2 and 3). These results clearly demonstrated that peroxynitritetreatment of prostaglandin H₂ synthase produced nitrotyrosine andinduced dimerization of PGHS-2 and formation of nitrotyrosine anddimerization of prostaglandin H₂ synthase were blocked by GSH.

[0068] Sheep placenta prostaglandin H₂ synthase form 2 containednitrated tyrosine as evidenced by the Western blot analysis withantibodies against nitrotyrosine (FIG. 6, Panel A). Immunoreactivity ofthe anti-tyrosine antibodies with the sheep placenta prostaglandin H₂synthase form 2 dramatically decreased after incubation with the primaryantibody solution containing nitrotyrosine. This result shows thatindeed the native prostaglandin H₂ synthase form 2 containednitrotyrosine formed in vivo.

[0069] Levels of nitrotyrosine in native proteins reflect in vivooxidant stress. In addition, the native sheep prostaglandin H₂ synthasewas found dimerized via a disulfide bond (FIG. 6, Panel B-3). In thepresence of βME, native sheep placenta prostaglandin H₂ synthase form 2was a monomer whereas in the absence of βME, the prostaglandin H₂synthase form 2 was a mixture of a monomer and a dimer (FIG. 6, PanelB-2 and B-2). Only the dimeric form of the prostaglandin H₂ synthaseform 2 was nitrated (FIG. 6, Panel B-3). These results show that thedimeric form of prostaglandin H₂ synthase form 2 was formed via adisulfide bond in vivo and only the dimeric form was nitrated.

[0070] Throughout this application, various publications, includingUnited States patents, are referenced by author and year and patents bynumber. Full citations for the publications are listed below. Thedisclosures of these publications and patents in their entireties arehereby incorporated by reference into this application in order to morefully describe the state of the art to which this invention pertains.

[0071] The invention has been described in an illustrative manner, andit is to be understood that the terminology which has been used isintended to be in the nature of words of description rather than oflimitation.

[0072] Obviously, many modifications and variations of the presentinvention are possible in light of the above teachings. It is,therefore, to be understood that within the scope of the describedinvention, the invention may be practiced otherwise than as specificallydescribed.

1. A method of assessing oxidant stress by measuring polymerization ofproteins.
 2. The method according to claim 1, wherein said measuringstep further comprises measuring nitrated, polymerized proteins.
 3. Themethod according to claim 2, wherein said measuring step includesmeasuring polymerized proteins selected from the group consistingessentially of polymerized prostaglandin H₂ synthase,nitrated-polymerized prostaglandin H₂ synthase, polymerized cytochromec, nitrated-polymerized cytochrome c, 30 kDa cytochrome c, nitrated 30kDa cytochrome c, 45 kDa cytochrome c, and nitrated 45 kDa cytchrome c.4. A marker for oxidant stress comprising a polymerized protein.
 5. Themarker according to claim 4, wherein said protein is nitrated.
 6. Themarker according to claim 5, wherein said marker is selected from thegroup consisting essentially of polymerized prostaglandin H₂ synthase,nitrated-polymerized prostaglandin H₂ synthase, polymerized cytochromec, nitrated-polymerized cytochrome c, 30 kDa cytochrome c, nitrated 30kDa cytochrome c, 45 kDa cytochrome c, and nitrated 45 kDa cytchrome c.7. The marker according to claim 4, wherein said marker is a disulfidebonded polymerized protein.
 8. The marker according to claim 7, whereinsaid marker is a nitrated disulfide bonded polymerized protein.
 9. Themarker according to claim 8, wherein said marker is selected from thegroup consisting essentially of disulfide bonded polymers ofprostaglandin H₂ synthase and nitrated, disulfide bonded polymers ofprostaglandin H₂ synthase.
 10. A kit for use in assessing oxidantstress, said kit comprising an assay for detecting polymerized proteins.11. The kit according to claim 10, wherein said assay further includesmeans for detecting nitrated polymerized proteins.
 12. The kit accordingto claim 10, wherein said assay further includes means for detecting theformation of disulfide bonded polymerized proteins.
 13. The kitaccording to claim 10, wherein said assay further includes means fordetecting the formation of nitrated disulfide bonded polymerizedproteins.
 14. A method of assessing oxidant stress by measuringnitrization of cytochrme c.
 15. A method of assessing oxidant stress bymeasuring the formation of disulfide polymerized proteins.
 16. Themethod according to claim 15, wherein said measuring step includesmeasuring the formation of nitrated-disulfide polymerized proteins. 17.A method of measuring oxidizing power of oxidizing molecules bymeasuring the amount of oxidized glutathione (GS-SG) formed from reducedglutathione (GSH).
 18. The method according to claim 17, wherein theoxidizing molecule is peroxynitrate.
 19. A method of measuring quenchingpower of reducing agents against oxidizing power of oxidizing moleculesby measuring a dimerized molecule (oxidized) of a reducing agent formedfrom a monomer (reduced) of the reducing agent.
 20. The method accordingto claim 19, wherein the oxidizing molecule is peroxynitrite.
 21. Themethod according to claim 19, wherein the reducing agent is GSH.
 22. Amethod of lowering oxidant stress by administering to a patient aneffective amount of at least one reducing agent which increases GSHlevels in a pharmaceutically acceptable carrier.
 23. The methodaccording to claim 22, wherein said administering step includesadministering a reducing agent which is glutathione.
 24. The methodaccording to claim 22, wherein said administering step includesadministering an agent for preventing polymerization of proteins. 25.The method according to claim 22, wherein said administering stepincludes administering an agent for preventing nitration of proteins.26. The method according to claim 22, wherein said administering stepincludes administering an agent for preventing polymerization andnitration of proteins.
 27. The method according to claim 22, whereinsaid administering step includes administering an agent for preventingdisulfide bonded prostaglandin H₂ synthase dimer formation.
 28. Themethod according to claim 22, wherein said administering step includesadministering an agent preventing nitrated, disulfide bondedprostaglandin H₂ synthase dimer formation.
 29. A pharmaceuticalcomposition for lowering oxidant stress, said pharmaceutical comprisingan effective amount of a reducing agent which increases GSH levels forreducing oxidant stress and a pharmaceutically acceptably carrier. 30.The pharmaceutical according to claim 2 9, wherein said reducing agentis glutathione.
 31. The pharmaceutical according to claim 29, whereinsaid reducing agent prevents polymerization of proteins.
 32. Thepharmaceutical according to claim 29, wherein said reducing agentprevents nitration of proteins.
 33. The pharmaceutical according toclaim 29, wherein said reducing agent prevents nitrated-disulfideprostaglandin H₂ synthase dimer foundation.